Terpenes hydrocarbons such as alpha and beta santalenes have been produced via biochemical processes for example such as through genetically altered cells. These terpenes and the alcohol derived from them are major constituents of sandalwood oil and the alcohols are important perfumery ingredients typically obtained commercially through the distillation of the heartwood of Santalum species (e.g., Sandalwood). Examples of such alcohols include α-sinensol, β-sinensol, α-santalol, β-santalol, α-trans-bergamotol and epi-β-santalol. Although new biochemical pathways have been developed, including genetically engineered cells, to generate the terpene hydrocarbons, it is desirable to find a biochemical pathway to generate and produce the alcohols derived from the santalenes. It is further desirable to use a biochemical pathway to not only generate such alcohols but it is further desirable to selectively produce, via a biochemical pathway, cis-isomers of the alcohols such as iso-α-sinensol, iso-β-sinensol, (Z)-α-santalol, (Z)-β-santalol, (Z)-α-trans-bergamotol and (Z)-epi-β-santalol.
Cytochrome P450s represent a family of enzymes of oxidases. P450s commonly catalyze a monooxygenase reaction. Cytochrome P450 enzymes are classified into families and subfamilies based on the amino acid sequences homology. Members of a same subfamily share over 55% amino acid sequence identity and have usually similar enzymatic activities (substrate and/or product selectivity). CYP71AV1 (NCBI accession No ABB82944.1, SEQ ID No. 51 and 52) and CYP71AV8 (NCBI accession No ADM86719.1, SEQ ID No. 1 and 2) are two members of the CYP71AV sub-family and shares 78% sequence identity. CYP71AV1 has previously been shown to oxidize amorphadiene (Teoh et al, FEBS letters 580 (2006) 1411-1416). CYP71AV8 has previously been shown to oxidize (+)-valencene, germacrene A and amorphadiene (Cankar et al, FEBS Lett. 585(1), 178-182 (2011)).
Processes using engineered cells have been reported that use terpene synthases to catalyze the production of a diterpene or sesquiterpene. The diterpenes or sesquiterpenes were further processed using a cytochrome P450 polypeptide to catalyze the hydroxylation, oxidation, demethylation or methylation of the diterpene or sesquiterpene produced by the cell.